Bone Marrow Vascular Niche Research Articles

Transcriptome Profiling of Glycosylation Genes Defines Correlation with E-Selectin Ligand Expression and Clinical Outcome in AML

E-selectin (E-sel) is a cell adhesion glycoprotein that is expressed on endothelial cells and has been implicated in therapeutic resistance. In most myeloid leukemias, leukemic blasts express E-sel ligands (EsL), which contain the glycan epitope of the carbohydrate sialyl Lex (sLex). This expression increases the likelihood of adhesion to vascular endothelial cells and facilitates sequestration in the bone marrow vascular niche, leading to cell adhesion-mediated drug resistance and poor clinical outcome. E-sel antagonists like uproleselan, interrupts leukemic cell homing to the vascular niche, increases susceptibility to cytotoxic and targeted therapies and can be potent adjuncts to therapeutics. Recent data demonstrated a correlation between leukemic cell surface levels of EsL and response to uproleselan, linking EsL expression to response. We questioned whether transcriptome profiling of EsL-forming glycosylation genes can be used to identify elevated EsL expression in patients with acute myeloid leukemia (AML), and subsequently which patients might best respond to uproleselan. RNA-seq data from patients treated in COG AAML1031 (N = 1,074) was available for evaluation. We examined transcriptome expression of 24 genes that code for enzymes involved in glycosylation of EsL. All analyses were performed in R. Cox proportional hazards models were generated using the survival package. Multidimensional flow cytometry (MDF) was used to detect cell surface EsL expression by two techniques: direct binding of an E-sel/hIg, PE labeled chimera, and the anti-sLex antibody HECA-452. Seven of the 24 genes examined had minimal expression (mean <1 TPM) and were excluded from further analysis. The remaining 17 were variably expressed (Fig. 1A). To assess association of expression with outcome, univariate Cox models for overall survival (OS) were generated, using gene expression as a continuous coefficient (N = 1,061). Of the 17 genes, 7 were significantly associated with increased risk (p < 0.05, Fig. 1B). ST3GAL4 and FUT7 were targeted for further evaluation, as they directly synthesize sLex (Fig. 1C), and were significantly associated with adverse outcome (HR = 1.013, p < 0.0001, and HR = 1.023, p < 0.0001, respectively). Patients highly expressing FUT7 (highest quartile of expression) had significantly worse outcome than low expressors (lowest 3 quartiles of expression), with a 5-year OS of 50.3% vs. 68.3% (p < 0.0001, Fig. 1D). Similarly, those with high ST3GAL4 expression had a 5-year OS of 51.3%, compared to 68.1% for low expressors (p < 0.0001, Fig. 1D). A subset of patients highly expressed both genes (ST3GAL4 and FUT7 high; SFhigh, N = 132). Compared to patients that did not highly express either gene (SFlow), these individuals had particularly adverse survival (45.8% OS vs 71.0% OS, p < 0.0001). Patients with one of two high expressing genes (SFinter) had a 5-year OS of 55.5%, illustrating what may be a compounding unfavorable impact conferred on survival (Fig. 1E). Further investigation of clinical characteristics within these 3 groups revealed that 71.5% of infants <1 year were SFlow, with only 4.66% in SFhigh. In addition, CBF-AML was greatly underrepresented in SFhigh, with 97% of both t(8;21) and inv(16) patients in SFlow, and 0% in SFhigh. To verify surface protein expression of the two genes, leukemic specimens from SFhigh patients (N = 10) and SFlow patients (N = 10) underwent cell surface expression evaluation of glycosylated EsL using two MDF assays. SFlow patients had low or undetectable levels of cell surface EsL by both assays, whereas SFhigh patients had significantly higher expression of EsL (p < 0.001, Fig. 1F). This suggests a strong correlation between transcriptome measurements of EsL glycosylation genes and cell surface glycosylation levels of EsL. In summary, we have shown that multiple genes involved in the glycan synthesis of EsLs are highly expressed in pediatric AML. Two of these genes, ST3GAL4 and FUT7, are associated with poor outcome. Additionally, high expression of these genes is detectable at the transcript level and associated with cell surface EsL expression. These genes provide novel targets for overcoming drug resistance induced by the tumor microenvironment, and lend support for the use of EsL glycosylation genes as predictive biomarkers. These data also confirm the importance of E-sel in disease progression in AML and its potential as a therapeutic target. Figure 1 Disclosures Pardo: Hematologics, Inc: Employment. Eidenschink Brodersen:Hematologics, Inc: Employment. Magnani:GlycoMimetics Inc: Employment, Equity Ownership. Fogler:GlycoMimetics Inc: Employment, Equity Ownership. Loken:Hematologics, Inc: Employment, Equity Ownership.

The Vasculopathy in the Bone Marrow Microenvironment of Humanized Sickle Cell Mice Is Reversible By Blood Transfusion

Sickle cell disease (SCD) is a monogenic red cell disorder with high morbidity and mortality due to significant end-organ disease. Here we investigated for the first time the impact of SCD on the bone marrow (BM) vascular niche, which is critical for hematopoiesis. In SCD mice, we find a disorganized and structurally abnormal BM vasculature with highly tortuous arterioles and fragmented sinusoidal vessels, the latter filled with aggregates of sickle erythroid and myeloid cells. By in vivo imaging, sickle and control red blood cells exhibit significantly slow intravascular flow speeds in sickle cell BM but not in control BM. These abnormalities lead to increased ROS production in expanded erythroblast populations, and to up-regulation in the BM of HIF-1a and key growth factors required to support angiogenesis, e.g. VEGF-A, Ang1, Ang2. Moreover, increased levels of VCAM-1, a marker for vascular dysfunction, are detected. SCD also impacts the BM perivascular niche, i.e. the number of CXCL12-abundant reticular cells, is markedly decreased, and is associated with mobilization of hematopoietic stem/progenitor cells as well as mature leukocytes to the periphery. Strikingly, the distorted vascular network is completely reversed by blood transfusion, which highlights the plasticity of the BM vascular niche. Thus, we provide molecular and cellular features of the pathologic BM microenvironment, which suggest that the local hypoxia-induced angiogenic network combined with inflammatory vascular dysfunction is responsible for the vasculopathy. In addition, the findings may serve to develop new biomarkers and therapeutic targets of BM end-organ damage. Figure Disclosures No relevant conflicts of interest to declare.

PEDF Can Rescue the Inhibition of Injured Endothelial Cells on Hematopoietic Stem Cell Expansion Ex Vivo

Introduction: Endothelial cells (ECs) provide a fertile niche for hematopoietic stem cell (HSC) maintenance, differentiation, and migration.Several studies have indicated that bone marrow (BM) vascular niche was impaired after HSC transplantation and severely inhibited hematopoietic reconstruction. Pigment epithelium-derived factor (PEDF) is an important potential cytoprotection and therapeutic agent for injured cells. The direct role of the injured endothelial cells on hematopoietic stem cells and whether PEDF has protective effect in this system remain unknown. This study aims to observe the influence of enjured ECs on HSCs and to explore the role of PEDF in endothelial-HSC coculture system. Methods: Injury of Endothelial cells by two important preparative regimenconditioning radiation and Busulfan respectively was evaluated with CCK8 assay. The expression of endothelial tight junctions(TJs),adherent junctions related molecules and endothelial to Mesenchymal Transition molecules such as ZO-1, Occludin,VE-cadherin, ICAM, α-SMA, CD31 and VCAM were detected by RT-qPCR, flow cytometry, immunofluorescence and western blot. The effects of injured endothelial cells on HSC self-renewal, differentiation, cell cycle and apoptosis were evaluated by flow cytometry, photography, viable cell count and clone formation assay. Hematopoiesis regulation factors SCF, IL-6, TGF-β and TNF-α were detected by ELISA. The protective effect of PEDF was also explored. Results: Both radiation and Busulfan could decrease cell viability of endothelial cells. The expression level of ZO-1, Occludin, VE-cadherin, ICAM, CD31 and VCAM were decreased and α-SMA was increased when EC exposed to radiation or Busulfan suggesting endothelial activation, impaired EC permeability and endothelial to Mesenchymal Transition after EC injured. Compared with normal endothelial cells and hematopoietic stem cell co-culture group, the HSC% of injured endothelial cells and hematopoietic stem cells co-cultured group were significantly decreased, the cell colony formation ability was decreased, the proportion of mature cells increased, and the damage of endothelial cells could not maintain the characteristics of HSC, weakened the self-renewal and multidirectional differentiation potential of HSC and promoted the maturation of HSC. After the administration of PEDF, endothelial to Mesenchymal Transition of EC was suppressed and the EC permeability was improved. Most importantly, the proportion of HSC was significantly increased, and the proportion of mature cells decreased in the coculture system. Conclusion: Injured endothelial cells can inhibit proliferation of hematopoietic stem cells, self-renewal and promote HSC differentiation. PEDF could ameliorate endothelial injury and promote HSC expansion by suppressing endothelial-mesenchymal transition and protecting TJs and AJs. Disclosures No relevant conflicts of interest to declare.

KRasG12D expression in the bone marrow vascular niche affects hematopoiesis with inflammatory signals

The bone marrow (BM) niche is an important milieu where hematopoietic stem and progenitor cells (HSPCs) are maintained. Previous studies have indicated that genetic mutations in various components of the niche can affect hematopoiesis and promote hematologic abnormalities, but the impact of abnormal BM endothelial cells (BMECs), a crucial niche component, on hematopoiesis remains incompletely understood. To dissect how genetic alterations in BMECs could affect hematopoiesis, we have employed a novel inducible Tie2-CreERT2 mouse model, with a tdTomato fluorescent reporter, to introduce an oncogenic KRasG12D mutation specifically in the adult endothelial cells. Tie2-CreERT2;KRasG12D mice had significantly more leukocytes and myeloid cells in the blood with mostly normal BM HSPC populations and developed splenomegaly. Genotyping polymerase chain reaction revealed KRasG12D activation in BMECs but not hematopoietic cells, confirming that the phenotype is due to the aberrant BMECs. Competitive transplant assays revealed that BM cells from the KRasG12D mice contained significantly fewer functional hematopoietic stem cells, and immunofluorescence imaging showed that the hematopoietic stem cells in the mutant mice were localized farther away from BM vasculature and closer to the endosteal area. RNA sequencing analyses found an inflammatory gene network, especially tumor necrosis factor α, as a possible contributor. Together, our results implicate an abnormal endothelial niche in compromising normal hematopoiesis.

6091Chronic heart failure is associated with inflammatory deterioration of the bone marrow vascular niche

Abstract Introduction Inflammation plays a crucial role in many aspects of cardiovascular disease. Particularly, acquired mutations of hematopoietic stem cells (HSC) leading to clonal expansion of inflammatory cells (CHIP) are increased with age and are associated with an enhanced risk of cardiovascular disease. The bone marrow (BM) vascular niche plays a crucial role in maintenance and regulation of HSC functions. Previous studies in mice showed the reduction of a specific Endomucin-high (H-type) endothelial cells (EC) subpopulation by aging. However, the impact of cardiovascular disease is unclear. Therefore, we aimed to investigate the effects of age and heart failure (HF) on the vascular BM cell composition in mice and humans. Methods and results Aging mice showed an age-dependent decrease of type H (Emcn-high) BM ECs (p=0.004), whereas the BM frequencies of type L (Emcn-low) ECs did not significantly differ (P=0.18). Importantly, we also observed a marked reduction of type H EC in chronic ischemic mice (P=0.016 vs. sham) indicating that chronic ischemic HF induces similar alterations of the vascular stem cell niche. Importantly, type H ECs were also significantly reduced in ischemic HF patients (n=16) compared with control subjects (n=8; P=0.0003). To gain insights into the mechanisms underlying the changes in the vascular niche, we performed single cells RNA sequencing of human BM ECs. These studies confirmed the decrease in Emcn-expressing ECs in ischemic HF patients, which was accompanied by significantly increased expression of inflammatory genes, including IL1b (P<0.0001 vs. control). Inflammatory EC phenotypes and IL1b expression in HF could be further confirmed at protein level using cytospin immunostainings. Finally, we comprehensively evaluated phenotype-associated differences in the bone marrow plasma proteomes of healthy individuals (n=19) and patients with chronic ischemic (n=22) and non-ischemic (n=19) HF, using proximity extension assays. Here, we identified 182 proteins significantly differentially regulated in CHF versus CTRL. Among the top upregulated proteins the BM environment of patients with CHF showed a striking enrichment of inflammatory and ECM remodeling components. Conclusions Our data show for the first time an impact of chronic heart failure on the bone marrow vascular niche in humans. These changes seem to be strongly associated with increased inflammatory response and bone matrix remodeling in CHF. Specifically, the induction of the inflammatory cytokine IL1b may contribute to the disturbed phenotype suggesting that inhibition of IL1b (e.g. by canakinumab) may be used as a novel strategy to prevent or reverse the deterioration of the vascular BM niche.

P1514Evaluation of endothelial progenitor cells as cardiovascular prognostic biomarkers in hemodialysis patients

Abstract Background Endothelial cells arise from endothelial progenitor cells (EPCs), which reside in bone marrow vascular niches, and are classified according to specific functional differences. Oxidative stress and inflammation lead to endothelial dysfunction that is a critical event in the initiation and progression of atherosclerotic plaques. Patients with chronic renal failure frequently show endothelial dysfunction and are at a greatly increased risk of developing atherosclerosis. Owing to their ability to partially restore vascular damage, both the number and functional changes in EPCs may be considered as useful prognostic biomarkers of cardiovascular events in hemodialysis (HD) patients. Purpose This study investigated EPC features and several biomarkers of systemic vascular inflammation and carotid atherogenesis in hemodialysis (HD) patients. Methods We studied 104 HD patients (males=55 and females 49, mean age: 51±12 years) and 40 healthy controls (males=20/females=20; mean age: 52±11 years). Isolated EPCs were cultured in the fibronectin-covered culture dishes and counted. EPC markers were studied by flow cytometry (FACS) by using anti-CD34, anti-CD133 and anti-vascular endothelial growth factor receptor 2 (VEGFR-2) antibodies. Serum levels of intercellular cell adhesion molecule (ICAM), vascular cell adhesion molecule (VCAM), IL-6, TNF-α, and asymmetric dimethyl-arginine (ADMA) were measured by ELISA method. Carotid intima-media thickness (CIMT) and ratio (CIMR) were also evaluated. Results In our experimental conditions, EPC number was decreased in HD patients when compared to controls (p<0.01). Expression of CD34 was significantly lower in the HD group (p<0.01). Interestingly, EPCs were significantly inversely correlated with serum TNF-α levels in HD patients (r=−0.388, p<0.01) but not in the control group (r=−0.133, p=NS). Furthermore, there was an inverse association between VEGFR-2 positive cells and serum TNF α levels (r=−0.401, p=0.002) in HD patients. In HD patients, there was a positive correlation between ICAM levels with CIMT (r=0.405, p=0.03) and CIMR was positively correlated with VCAM levels (r=0.377, p=0.01) and ADMA (r=0.233, p<0.05). Conclusions Our study shows that EPC number was decreased in HD patients and it was associated with systemic inflammation. TNF-α could activate inhibitory actions on EPC in HD patients. A significant relationship was present between ICAM/VCAM and carotid atherosclerosis, while this was not evident with EPC number. These pathogenic mechanisms can contribute to the high incidence of cardiovascular diseases in HD patients. However, further larger studies should investigate this working hypothesis.

1015 - INHIBITION OF ENDOTHELIAL MTOR DRIVES HEMATOPOIETIC STEM CELL AGING

Aging leads to a decline in hematopoietic stem cell (HSC) function. While some of these age-related changes reflect intrinsic alterations within HSCs, recent findings suggest that signals from the bone marrow (BM) microenvironment, in particular the BM vascular niche, might play a crucial role in regulating HSC aging. To this end, we recently discovered that aging of BM endothelial cells (BMECs) leads to an altered molecular crosstalk between the BMEC niche and HSCs that instructs young HSCs to behave as aged HSCs. Here, we demonstrate that aging leads to a decrease in mTOR signaling within BMECs that underlies the age-related impairment of their niche activity. In support of this idea, our findings reveal that pharmacological inhibition of mTOR signaling using Rapamycin, a widely regarded rejuvenating agent and clinically utilized immunosuppressant, has deleterious effects on the hematopoietic system, in part due to its adverse impact on endothelium by inhibition of both mTORC1 and mTORC2 signaling. To formally determine whether EC-specific inhibition of mTOR signaling can influence hematopoietic aging, we conditionally deleted mTOR in ECs (mTOR(ECKO)) of young mice and observed that their BMECs and HSCs displayed all the phenotypic and functional attributes of an aged hematopoietic system. Gene expression profiling of HSCs isolated from young mTOR(ECKO) mice revealed that their transcriptome resembled aged HSCs, both at steady state and following transplantation. Notably, during serial transplantations, these aging-like phenotypic and functional HSC alterations are only maintained in an mTOR(ECKO) microenvironment confirming the role of EC-derived instructive signals in governing HSC aging. Furthermore, analysis of mTOR(ECKO) mice revealed a pro-aging factor wherein its genetic deletion led to a preservation of HSC function in aged mice. These studies have begun to lay the framework for the development of therapeutic modalities designed to restore the functional properties of the aged hematopoietic system to youthful levels and improve overall health span.

Angiocrine signals regulate quiescence and therapy resistance in bone metastasis.

Bone provides supportive microenvironments for hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) and is a frequent site of metastasis. While incidences of bone metastases increase with age, the properties of the bone marrow microenvironment that regulate dormancy and reactivation of disseminated tumor cells (DTCs) remain poorly understood. Here, we elucidate the age-associated changes in the bone secretome that trigger proliferation of HSCs, MSCs, and DTCs in the aging bone marrow microenvironment. Remarkably, a bone-specific mechanism involving expansion of pericytes and induction of quiescence-promoting secretome rendered this proliferative microenvironment resistant to radiation and chemotherapy. This bone-specific expansion of pericytes was triggered by an increase in PDGF signaling via remodeling of specialized type H blood vessels in response to therapy. The decline in bone marrow pericytes upon aging provides an explanation for loss of quiescence and expansion of cancer cells in the aged bone marrow microenvironment. Manipulation of blood flow — specifically, reduced blood flow — inhibited pericyte expansion, regulated endothelial PDGF-B expression, and rendered bone metastatic cancer cells susceptible to radiation and chemotherapy. Thus, our study provides a framework to recognize bone marrow vascular niches in age-associated increases in metastasis and to target angiocrine signals in therapeutic strategies to manage bone metastasis.

Dysregulated megakaryocyte distribution associated with nestin+ mesenchymal stem cells in immune thrombocytopenia

AbstractImpaired megakaryocyte (MK) maturation and reduced platelet production are important causes of immune thrombocytopenia (ITP). However, MK distribution and bone marrow (BM) niche alteration in ITP are unclear. To investigate the maturation and distribution of MKs in the BM niche and examine the components of BM niche regulation of MK migration, BM and peripheral blood were obtained from 30 ITP patients and 28 healthy donors. Nestin+ mesenchymal stem cells (MSCs) and CD41+ MKs were sorted by fluorescence-activated cell sorting. The components of the BM niche and related signaling were analyzed via immunofluorescence, flow cytometry, enzyme-linked immunosorbent assay, reverse transcription polymerase chain reaction, and western blot analysis. The number of MKs in the BM vascular niche was reduced in ITP. Moreover, the concentrations of CXCL12 and CXCR4+ MKs in the BM were decreased in ITP. Further investigation demonstrated that nestin+ MSCs and CXCL12 messenger RNA (mRNA) in nestin+ MSCs were both reduced whereas the apoptosis of nestin+ MSCs was significantly increased in ITP. Sympathetic nerves, Schwann cells, the proportion of β3-adrenoreceptor (β3-AR)+ nestin+ MSCs, and β3-AR mRNA in nestin+ MSCs were all markedly reduced in ITP. Moreover, matrix metalloproteinase 9, vascular endothelial growth factor (VEGF), and VEGF receptor 1 were significantly reduced in ITP. Our data show that impaired MK distribution mediated by an abnormal CXCL12/CXCR4 axis is partially involved in reduced platelet production in ITP. Moreover, sympathetic neuropathy and nestin+ MSC apoptosis may have an effect on the alterations of BM CXCL12 in ITP.

Role of Vascular Niche of Bone Marrow in the Development of MLL-AF9 Induced Acute Myclaid Leukemia

To investigate the role of bone marrow vascular niche in the development of MLL-AF9 acute myeloid leukemia (AML). Transplantation experiments were performed to establish non-radiated MLL-AF9 AML model; the half-bone immunofluorescence staining and tile-scan imaging of two-photon confocal microscopy were used to obtain the data of 3 main bone marrow niche cells; flow cytometry analysis was performed to characterize leukemia cells in different anatomical sites. In the early stage of MLL-AF9 AML, the proportion of leukemia cells in the metaphysis of the femur was significantly higher than that in the diaphysis. The detection of apoptosis and proliferation rate of leukemia cells showed that the percentage of leukemia cells in metaphysis significantly decreased, and the proliferation (S/G2/M phase) was also significantly more active. These different features of leukemia cells may relate with different bone marrow microenvironment. The image data of 3 major components of bone marrow niche (endothelial cells, endosteum, megakaryocytes) showed different distribution of blood vessels in metaphysis and diaphysis. Furtherly comparing the spatial distance between leukemia cells and endothelial cells, endosteum, megakaryocytes indicated that leukemia cells are closer to the blood vessels, suggesting the important role of blood vessels in the development of leukemia. Glucose uptake assays and intracellular ROS detection showed that the supportive role of blood vessels for leukemia cells did not related with nutrient metabolism pathway. The vascular niche plays an important role in the development of leukemia, and does not relate with the transport of nutrients and the elimination of metabolic waste, instead, which may relate with perivascular cytokines or other vascular functions.

Myocardial infarction induces bone marrow megakaryocyte proliferation, maturation and platelet production

Platelet, apart from its classic role of homeostasis, serves also as a crucial immune cell component that contributes to the aggravation of atherosclerosis. It has been reported that myocardial infarction (MI) triggers leukocytosis in the bone marrow and spleen, which accelerates post-MI atherosclerosis. However, it remains unclear whether thrombopoiesis is also enhanced after MI. Here, using flow cytometry and bone marrow whole-mount immunofluorescence staining combined with three-dimensional (3D) reconstruction, we for the first time demonstrated an enhanced thrombopoiesis and megakaryopoiesis in a mouse model of coronary artery ligation as a mimic of MI. We showed that MI leads to increasing number of peripheral platelets, as well as elevating number and larger size of bone marrow MKs. We also observed more proplatelets and fragmented MKs, and a closer spatial distribution of MK populations to the bone marrow vascular niche after MI. This study provides direct evidence that MI induces bone marrow megakaryocyte proliferation, maturation and platelet production. It opens a new scope that targeting platelet production might become a novel therapeutic approach for attenuating post-MI atherosclerosis.

Inhibition of Semaphorin 3A Signaling Promotes Regeneration of Hematopoietic Stem Cells and Their Bone Marrow Vascular Niche

Radiation injury damages both the bone marrow hematopoietic stem cell and its supportive endothelial niche. In response to radiation stress, bone marrow endothelial cells (BM ECs) upregulate expression of a gene, Sema3A, which encodes for the secreted protein, Semaphorin 3A (SEMA3A), and its receptor, Neuropilin 1 (Nrp1). Commensurate with this, we observed a substantial increase in SEMA3A protein levels in the BM of mice and in NRP1 expression on VE-cadherin+ BM ECs following 500 cGy total body irradiation (TBI) (p<0.001, p<0.0001). We also found that treatment of irradiated primary murine BM ECs with SEMA3A significantly increased BM EC apoptosis (p<0.01). Based on these results, we hypothesized that SEMA3A may be a negative regulator of BM vascular recovery following injury and that inhibition of SEMA3A/NRP1 signaling might accelerate BM vascular niche recovery following irradiation. In keeping with this hypothesis, we found that a single systemic administration of 2 µg SEMA3A (IV) to C57BL/6 mice following 500 cGy TBI significantly increased BM EC apoptosis in vivo at 24 hours and also markedly decreased BM vascular integrity at 24 hours as measured by Evans Blue Dye extravasation in the BM (p<0.01, p<0.01). Interestingly, SEMA3A administration (IV, every other day) also significantly suppressed BM hematopoietic regeneration with decreased BM cell counts and BM ckit+sca-1+lin- (KSL) progenitor cell recovery at day +10 compared to irradiated, control mice (p=0.04, p=0.001).In contrast, systemic administration of anti-NRP1 antibody (10 µg, IV every other day), which blocks SEMA3A binding to NRP1 on BM ECs, accelerated regeneration of the sinusoidal BM vasculature at day +10 following 500 cGy TBI compared to irradiated controls. Concordant with these findings, anti-NRP1 treatment increased the recovery of peripheral blood WBCs, neutrophils, and BM KSL cells, compared to irradiated controls (p=0.002, p=0.04, p=0.04). Competitive repopulation assays confirmed that anti-NRP1 treatment increased the recovery of long-term HSCs capable of 20-week multilineage repopulation in congenic mice (p=0.03 for % donor CD45.1+ cell engraftment at 20 weeks). Importantly, anti-NRP1 treatment also markedly improved 60-day survival of irradiated mice from 17% (2/12 controls) to 83% (10/12 anti-NRP1) following 800 cGy TBI.In order to confirm the role of SEMA3A/NRP1 signaling in BM ECs in regulating the BM vascular and hematopoietic response to injury, we generated mice with tamoxifen-inducible, EC - specific deletion of Nrp1 (VE-Cad-Cre-ERT2;Nrp1 fl/fl mice). Adult VE-Cad-Cre-ERT2;Nrp1 fl/fl mice were viable and had no baseline hematologic phenotype. However, following 500 cGy TBI, VE-Cad-Cre-ERT2;Nrp1 fl/fl mice displayed accelerated BM vascular regeneration at day +10 compared to irradiated control mice. Furthermore, VE-Cad-Cre-ERT2;Nrp1 fl/fl mice demonstrated significantly increased peripheral blood WBCs, neutrophils, and BM KSL cells at day +10 following radiation injury (p<0.001, p=0.002, p=0.002). These data suggest that the SEMA3A-NRP1 pathway is an autocrine signaling mechanism that negatively regulates BM vascular niche recovery following myelosuppressive irradiation. Targeted inhibition of SEMA3A-NRP1 signaling in BM ECs has therapeutic potential to accelerate both BM vascular niche regeneration and hematopoietic reconstitution following myelosuppression. DisclosuresNo relevant conflicts of interest to declare.

Inhibition of Endothelial mTOR Drives Hematopoietic Stem Cell Aging

Aging leads to a drastic decline in hematopoietic stem cell (HSC) function. In addition to a loss of their self-renewal potential, old HSCs exhibit a myeloid biased differentiation and an increased propensity to develop hematologic malignancies. While some of these age-related changes reflect cell-intrinsic alterations within HSCs, recent findings suggest that signals from the bone marrow (BM) microenvironment, in particular the BM vascular niche, might play crucial roles in regulating HSC aging. In support of this idea, we recently discovered that physiological aging of BM endothelial cells leads to an altered molecular crosstalk between the endothelial niche and HSCs that instructs young HSCs to behave as aged HSCs. We have previously demonstrated that AKT/mTOR activation within young BM endothelial cells supports balanced HSC self-renewal and differentiation. Based on these observations, we hypothesized that deficient AKT/mTOR signaling within endothelium will deprive the hematopoietic system of proper endothelial-derived instructive signals resulting in HSC aging phenotypes. We have generated preliminary data demonstrating that aged endothelial cells, in contrast to aged HSCs, display decreased AKT/mTOR signaling. Furthermore, our data indicates that pharmacological inhibition of mTOR signaling using Rapamycin, a widely regarded rejuvenating agent and clinically utilized immunosuppressant, has deleterious effects on the hematopoietic system, in part due to its adverse impact on endothelium by abolishing both mTORC1 and mTORC2 signaling, in homeostatic conditions and following myelosuppressive therapy (i.e. radiation and chemotherapy). To formally determine whether endothelial-specific inhibition of mTOR signaling can promote hematopoietic aging, we conditionally deleted mTOR in ECs (mTOR(ECKO)) of young mice and observed that HSCs from mTOR(ECKO) mice displayed all the phenotypic and functional attributes of an aged HSC including loss of cell polarity, increased DNA damage, myeloid biased output and loss of engraftment potential during serial transplantation. Gene expression profiling of HSCs isolated from young mTOR(ECKO) mice revealed that their intrinsic gene expression signature resembled aged HSCs, both at steady state and following transplantation. Notably, these aging-like phenotypic and functional HSC alterations are only maintained in secondary transplants that were performed in an mTOR(ECKO) microenvironment confirming the role of EC-derived instructive signals in governing HSC aging. Furthermore, we assessed the function of BM endothelial cells from mTOR(ECKO) mice and revealed severe defects in overall endothelial health which included alterations in proliferation, wound healing ability, and their metabolic profile that displayed a hyperglycolytic phenotype at steady state as well as increased glycolytic capacity under stress conditions, compared to control BMECs. Moreover, mTOR(ECKO) BMECs showed high respiratory capacity, thus phenocopying the metabolic profile of aged and tumor-associated BMECs. Furthermore, the BM microenvironment of mTOR(ECKO) mice manifested an increase in a NF-kB dependent inflammatory signature. Interestingly, chronic inflammation driven by NF-kB signaling is the leading cause of vascular dysfunction and aging. In summary, we demonstrate that deletion of mTOR signaling within young endothelium drives premature aging of HSCs and the hematopoietic system. Utilizing our model system, we have now identified candidate pro-aging and pro-rejuvenation factors that will lay the foundation for designing therapeutic strategies to rejuvenate the HSC microenvironment, thereby restoring the functional properties of the hematopoietic system to youthful levels and improving overall health. DisclosuresNo relevant conflicts of interest to declare.

Hematopoietic insults damage bone marrow niche by activating p53 in vascular endothelial cells

Hematopoietic stem cells (HSCs) are exposed to various insults such as genotoxic stress, inflammation, and infection, which have a direct effect. These insults deplete, cause a functional decline in, and promote HSC aging and transformation. However, the impact of hematopoietic insults on niche cells remains largely unknown. We have reported previously that p53 is activated in blood vessels by various stresses, including hypoxia, inflammation, and aging, and contributes to tissue dysfunction and metabolic abnormalities. We hypothesized that hematopoietic insults also affect the bone marrow (BM) vascular niche. Here, we demonstrate that p53 becomes activated in BM endothelial cells upon hematopoietic stresses such as irradiation and chemotherapeutic treatments. The conditional activation of p53 in VE-cadherin+ vascular niche cells by deleting Mdm2 induces the expression of p53 target genes specifically in vascular endothelial cells, resulting in the dilation and collapse of vascular endothelial cells and reductions in perivascular mesenchymal stromal cell numbers. Consequently, hematopoietic stem cells (HSCs) fail to maintain dormancy, mobilize to the periphery, and are depleted significantly. Our results indicate that various hematopoietic insults affect HSCs, not only directly, but also indirectly by altering vascular integrity, which is critical for perivascular niche formation and maintenance of HSCs.

RUNX transcription factors potentially control E-selectin expression in the bone marrow vascular niche in mice

AbstractAlthough the function of Runt-related (RUNX) transcription factors has been well characterized in leukemogenesis and regarded as an ideal target in antileukemia strategies, the effect of RUNX-inhibition therapy on bone marrow niche cells andr its impact on the engraftment of acute myeloid leukemia (AML) cells have largely been unknown. Here, we provide evidence suggesting the possible involvement of RUNX transcription factors in the transactivation of E-selectin, a member of selectin family of cell adhesion molecules, on the vascular endothelial cells of the mice bone marrow niche. In our experiments, gene switch-mediated silencing of RUNX downregulated E-selectin expression in the vascular niche and negatively controlled the engraftment of AML cells in the bone marrow, extending the overall survival of leukemic mice. Our work identified the novel role of RUNX family genes in the vascular niche and showed that the vascular niche, a home for AML cells, could be strategically targeted with RUNX-silencing antileukemia therapies. Considering the excellent efficacy of RUNX-inhibition therapy on AML cells themselves as we have previously reported, this strategy potentially targets AML cells both directly and indirectly, thus providing a better chance of cure for poor-prognostic AML patients.

Vascular E-Selectin Mediates Chemo-Resistance in Acute Myeloid Leukemia Initiating Cells Via Canonical Receptors PSGL-1 (CD162) and Hcell (CD44) and AKT Signaling

The vascular adhesion molecule E-selectin is a key component of the bone marrow (BM) vascular niche, awakening otherwise dormant hematopoietic stem cells (HSC) (Winkler et al., Nat Med 2012). Vascular niches also mediate malignant cell survival. We find E-selectin becomes upregulated on BM vasculature during leukemia and that adhesion of BM AML blasts to E-selectin promotes survival signaling. This is unique to E-selectin - not observed with adhesion to P-selectin, PECAM1, or integrin ligands ICAM1, VCAM1. In vivo we find absence (in SELE-/- mice) or therapeutic blockade of E-selectin by administration of E-selectin antagonist GMI-1271 to mice, induces 9-fold greater chemosensitivity in 11q23-rearranged AML Initiating Cells (LIC) to cytarabine therapy in vivo and doubles survival over that achieved by chemotherapy alone. Together these data support the Phase I/II clinical trial of GMI-1271 in combination with intensive chemotherapy to improve efficacy of therapy for AML (NCT02306291), and raise the further questions: 1) what are the AML LIC ligands interacting with E-selectin at the vascular niche and 2) what are the pathways initiated by E-selectin adhesion that mediate chemoresistance.Using murine models of AML generated by retroviral transduction of the 11q23 fusion oncogene MLL-AF9 into HSC, we find leukemic blasts rapidly upregulate E-selectin binding potential upon oncogenic transformation. To investigate if this is due to aberrant glycosylation facilitating the generation of de novo AML cell surface receptors, we generated AML from HSC knocked-out for the two canonical E-selectin receptors PSGL1 and CD44. Surprisingly we found although HSC from CD44-/-PSGL1-/- mice still bound to E-selectin (indicating HSC express additional E-selectin ligands), AML cells generated from these same HSCs no longer did. Thus the functional E-selectin ligands utilized by murine HSC and AML blasts differ.When transplanted into recipient mice CD44-/-PSGL1-/- AML engrafted with similar disease progression to matching wildtype AML but demonstrated ~100-fold greater sensitivity to high-dose cytarabine chemotherapy. When GMI-1271 was co-administered together with chemotherapy no further boost in chemosensitivity was observed in CD44-/-PSGL1-/- AMLs. This complements in vitro data showing E-selectin adhesion-mediated chemoresistance is lost when CD44 and/orPSGL1are absent in AML cells. Together suggesting that CD44 and/or PSGL1 are the key receptors involved in E-selectin-mediated pro-survival signaling.To understand the pro-survival signaling pathways involved, E-selectin antagonist GMI1271 or saline control were administered 5 days to mice with AML and BM KIT+ leukemic blasts sorted. RNA sequencing revealed only a small number (~200) of mRNA transcripts differentially regulated following therapeutic E-selectin blockade. Of note these included down-regulation in several components of the PI3K/AKT/NF-kB signaling pathway frequently involved in cell survival. Indeed AKT Ser473 and NF-kB p65 Ser536 were found to be rapidly phosphorylated within 15 minutes of E-selectin adhesion in AML blasts in vitro. This phosphorylation was specific to E-selectin adhesion and not observed with other vascular adhesion molecules tested suggesting that direct E-selectin-mediated AKT activation in AML cells at the vascular niche may be the potential mechanism driving vascular mediated chemoresistance.To confirm whether therapeutic blockade of E-selectin alone dampens intracellular AKT signaling in AML blasts, cohorts of mice with wildtype or matching CD44-/-PSGL1-/- AML were administered GMI-1271 for 5 days then BM AML blasts collected for signaling studies. Both the administration of E-selectin antagonist or absence of CD44 and/or PSGL-1 dampened AKTSer473 phosphorylation in BM AML blasts in vivo, correlating with their heightened sensitivity to chemotherapy .In summary, these findings suggest E-selectin ligands on specific CD44 and/or PSGL1 glycoforms are the predominant determinants through which the induction/amplification of chemoresistant pathways in AML blasts occur following vascular adhesion and further that these pathways are coupled to an early phosphorylation event of AKT - and finally explain potential mechanism how GMI-1271 administration is able to attenuate vascular-mediated signaling and restore susceptibility of AML to chemotherapy. DisclosuresMagnani:GlycoMimetics, Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Vascular endothelial cells take hematopoietic stem cells to school

The bone marrow vascular endothelial cell niche plays a critical role in regulating hematopoietic system aging by influencing the phenotype of hematopoietic stem cells.

Abstract 876: Imaging the interaction of leukemia and bone marrow microenvironment in murine model of ALL

Abstract Interactions of leukemia and the bone marrow (BM) microenvironment are known to play a key role in the survival and growth of leukemic cells, and we have shown that HIF-1α stabilization in BM stromal cells facilitates leukemia homing and progression (Chen et al. Blood 2012, 119:4971). Leukemic cells have been shown to hijack the homeostatic mechanisms of normal hematopoietic stem cells (HSCs) and take refuge within the BM niche. This mechanism is pivotal during chemotherapy and contributes to disease relapse. In this study, we aimed to characterize the time-dependent progression of BM hypoxia involving both acute lymphocytic leukemia (ALL) cells and components of the BM niche, using multiphoton intravital microscopy (MP-IVM) . We generated a transplantable, fluorescent leukemia model by retrovirally transducing C57Bl6-Ai14 murine BM cells that express red fluorescing tdTomato with the p190-Bcr/Abl oncogene (KG Harutyunyan et al, Blood 2014 124:2396). The resulting p190-Bcr/Abl tdTomato cells caused rapid development of ALL in non-irradiated C57Bl6 immunocompetent mice, manifested by infiltration of multiple organ and BM sites, followed by death within 14-18 days. We utilized Col2.3-GFPemd transgenic mice as recipients of leukemia to highlight the osteoblastic niche, and visualized vasculature by injection of TRITC-dextran. We showed the dynamic of homing and engraftment of ALL leukemic B cells (LBC) in OB-GFP recipient mice, with homing in the vicinity of blood vessels visualized by MP-IVM, followed by proliferation and leukemia expansion. This was accompanied by invasion of both vascular and osteoblastic components of BM microenvironment. Longitudinal assessment of hypoxia utilizing pimonidazole staining showed progressive development of BM hypoxia starting from Day 10 p.i., paralleling leukemia progression, despite the abundant vascularization of the BM. To assess the integrity of the BM vascular niche during leukemia progression the experimental mice were intravitally injected with low doses of an Alexa-Fluor-647-conjugated VE-cadherin antibody (MG. Poulos et al, Epub 2013 Sep 5) to visualize the number and morphology of the perfused vessels and to analyze the architecture of the hematopoietic compartment. We observed that at late stage of leukemia progression (day 12-14 p.i) BM vessels are disorganized as a result of expansion of leukemic B cells. Ongoing longitudinal imaging experiments will characterize the cellular origin of hypoxic niche cells and the dynamic of vasculature alteration in ALL. In summary, these findings demonstrate rapid development of intra-BM hypoxia that parallels leukemia progression and involves interactions between leukemia cells and BM niche cells, as well as disordered vasculature. Citation Format: Karine G. Harutyunyan, Saradhi Mallampati, Anna Zal, Mateusz Rytelewski, Michael C. Gutkin, Jason M. Butler, Tomasz Zal, Marina Konopleva. Imaging the interaction of leukemia and bone marrow microenvironment in murine model of ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 876. doi:10.1158/1538-7445.AM2017-876

Desialylation of Megakaryocytes Diminishes Platelet Production By Disrupting Megakaryocyte Adhesion, Migration and Proplatelet Formation in Chronic Immune Thrombocytopenia

Immune thrombocytopenia (ITP) is a common autoimmune disorder characterized by increased bleeding tendency and isolated thrombocytopenia. The precise pathogenesis of the decreased thrombopoiesis in chronic ITP (cITP) is poorly understood. Megakaryocytes (MKs) in cITP show impaired maturation and thrombopoiesis, which are correlated with numerous glycoproteins on the surface of MKs. Different types of sialoglycoproteins are expressed on the surface of megakaryocytes, including GPIbα and platelet endothelial cell adhesion molecule-1 (PECAM-1), both of which participate in megakaryocyte migration to the vascular niche in the bone marrow (BM) and in proplatelet formation. Desialylation has recently been identified a contributor to the pathogenesis of thrombocytopenia. Our previous study has demonstrated that desialylation of GPIbα is related to increased apoptosis and phagocytosis of platelets in cases of prolonged isolated thrombocytopenia after allogeneic hematopoietic stem cell transplantation (Zhang et al., J Hematol Oncol, 2015). Because MKs are heavily sialylated cells, we raised the question whether the desialylation of megakaryocytes contributes to the defective thrombopoiesis in patients with cITP through impaired MK migration, adhesion and proplatelet formation in the vascular niche.MK desialylation was analyzed by flow cytometry using lectins. Desialylated glycoproteins were measured using selective exo-enzymatic labeling. Protein expression, distribution and interaction were measured using the following techniques: immunofluorescence, flow cytometry, western blot and immunoprecipitation. cITP MKs exhibited increased β-galactose exposure compared to the control MKs, indicating excessive desialylation. Desialylation was correlated with decreased platelet production of MKs. We further explored the cause of desialylation and found that the sialidase NEU1 was over-expressed in MKs. Treatment with the sialidase inhibitor DANA ameliorated the loss of sialic acids. These results indicated that NEU1 contributed to the desialylation of MKs in cITP. Altered MK distribution in the BM niche was exhibited upon BM biopsy of cITP patients. The ratio of perivascular MKs was markedly decreased in cITP patients. Defective adhesion and transmigration behaviors were also discovered in desialylated cITP MKs. The motility of cITP MKs through stromal cell monolayers driven by stromal cell derived factor 1 (SDF1) was decreased. Adherence to fibronectin, collagen and fibrinogen was assessed, and desialylated cITP MKs exhibited an increase in adhesion with these macromolecules. Similar abnormalities were observed in the BM niche of ST6Gal1-/- mice, and treatment with ST6Gal1 and CMP-SA augmented the ratio of MKs in the BM vascular niche in ST6Gal1-/-mice, indicating that desialylation impaired the MK migration and adhesion. Additional experiments focused on which specific sialoglycoproteins are excessively desialylated. As detected by SEEL, PECAM-1 exhibited excessive desialylation in cITP MKs, which was related to impaired CXCR4 polarization in response to SDF1. Inhibition of sialidase using DANA partially restored this polarization, demonstrating that desialylation of PECAM-1 was responsible for the defect in MK migration to the vascular niche. Meanwhile, PECAM-1 desialylation was associated with GPIIb/IIIa overactivation, which correlated to the increased adhesion of MKs. This increased adhesion was reversed by the GPIIb/IIIa inhibitor lotrafiban, indicating that desialylated PECAM-1 contributed to the abnormal adhesion via overactivation of GPIIb/IIIa. Desialylation of GPIbα was found on the surface of MKs from cITP patients and was associated with abnormal microtubule formation and increased MK apoptosis through altered 14-3-3ζ distribution, which led to the impediment of proplatelet formation in the vascular niche.In conclusion, our results demonstrate that MKs are desialylated by NEU1 in cITP patients, and desialylated MKs present with defective migration towards the vascular niche, abnormal adhesion to the extracellular matrix and impaired proplatelet formation. Desialylation of PECAM-1 and GPIbα have been demonstrated to be responsible for these abnormal behaviors of MKs. Sialidase inhibitor shows an improvement in the thrombopoiesis of cITP MKs; therefore, our study implies a novel potential approach for the treatment of cITP. DisclosuresNo relevant conflicts of interest to declare.

Endothelial-specific inhibition of NF-κB enhances functional haematopoiesis

Haematopoietic stem cells (HSCs) reside in distinct niches within the bone marrow (BM) microenvironment, comprised of endothelial cells (ECs) and tightly associated perivascular constituents that regulate haematopoiesis through the expression of paracrine factors. Here we report that the canonical NF-κB pathway in the BM vascular niche is a critical signalling axis that regulates HSC function at steady state and following myelosuppressive insult, in which inhibition of EC NF-κB promotes improved HSC function and pan-haematopoietic recovery. Mice expressing an endothelial-specific dominant negative IκBα cassette under the Tie2 promoter display a marked increase in HSC activity and self-renewal, while promoting the accelerated recovery of haematopoiesis following myelosuppression, in part through protection of the BM microenvironment following radiation and chemotherapeutic-induced insult. Moreover, transplantation of NF-κB-inhibited BM ECs enhanced haematopoietic recovery and protected mice from pancytopenia-induced death. These findings pave the way for development of niche-specific cellular approaches for the treatment of haematological disorders requiring myelosuppressive regimens.